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Implementation of MALDI MS profiling and imaging

methods for the analysis of real crime scene fingermarks

BRADSHAW, Robert <http://orcid.org/0000-0003-1533-2166>, DENISON, N. and FRANCESE, Simona <http://orcid.org/0000-0002-1381-1262>

Available from Sheffield Hallam University Research Archive (SHURA) at:

http://shura.shu.ac.uk/15678/

This document is the author deposited version. You are advised to consult the publisher's version if you wish to cite from it.

Published version

BRADSHAW, Robert, DENISON, N. and FRANCESE, Simona (2017).

Implementation of MALDI MS profiling and imaging methods for the analysis of real crime scene fingermarks. Analyst, 142 (9), 1581-1590.

Copyright and re-use policy

See http://shura.shu.ac.uk/information.html

Sheffield Hallam University Research Archive

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Implementation of MALDI MS Profiling and Imaging methods for the analysis of real crime scene fingermarks.

R. Bradshaw1, N. Denison2 and S. Francese1*

1

Centre for Mass Spectrometry Imaging, Biomolecular Research Centre, Sheffield Hallam University,

Sheffield, UK, S1 1WB

2

Identification Services Yorkshire and the Humber Region, West Yorkshire Police, Wakefield, UK

WF27UA.

* corresponding author: Simona Francese, email: [email protected]

Abstract

In the study presented here, four examples of crime scene fingermarks analysed by Matrix

Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI), in

collaboration with the local Police Force, are reported. These marks are associated to high

profile crimes such as drug dealing, murder and harassment. Following the application of

forensic enhancement techniques by the CSI or the crime lab, one of the marks could be

directly analysed on the surface of deposition and the others were analysed as primary lifts. In

one case, no physical or molecular information was obtained whereas in two cases both ridge

detail (albeit not useful for identification purposes) and molecular information could be

retrieved. In one case, the intelligence gathered around the suspect's state of mind is, to date

the most accomplished demonstration of the benefits and the operational feasibility MALDI

MS based methods. These four casework examples are to be considered a first insight into the

limitations, benefits and feasibility of MALDI MS based methods in the field; due to the

extreme variability in the state of the mark, comprehensive evaluation of these aspects can

only be undertaken upon the analysis of a large number of crime scene marks. However, this

work does represent a significant advancement, compared to previous published work, as it

demonstrates operational feasibility, with different levels of success, despite uncontrollable,

unknown and unaccountable environmental and donor variability.

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Introduction

Since 2008, several examples of chemical imaging techniques have been reported in the

literature for application to latent fingermark analysis. The interest of the analytical

community in developing methods to detect and map the molecular make up of latent

fingermarks has the potential to be a real game changer with respect to more informed

investigations and judicial debates. Amongst the analytical techniques, Desorption

Electrospray Ionisation (DESI) was the first mass spectrometric technique to be reported as

suitable for latent fingermark imaging1, followed by Surface Assisted Laser Desorption

Imaging (SALDI)2, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Profiling

and Imaging (MALDI MSP and MSI)3, Secondary Ion Mass Spectrometry (SIMS)4 and

Direct Analysis in Real Time (DART)5, with SALDI and DART being mostly used for

chemical analysis rather than imaging purposes. Amongst the spectroscopic techniques,

Raman Imaging6 and FT-IR7 or ATR FT-IR Imaging8 have also been reported to be suitable for latent fingermark analysis in laboratory settings. Out of the above mentioned techniques,

only MALDI, SIMS and ATR FT-IR were selected by the Centre of Applied Science and

Technology, Home Office UK, to be included within the Fingermark Visualisation Manual9

as techniques having potential to be used in casework. Of these technologies, MALDI MSP

and MSP have arguably demonstrated superiority with regards to integration and

compatibility with the fingermark enhancement workflow. Furthermore, in the present paper,

it will be demonstrated for the first time that these techniques have reached a level of

maturity like no other mass spectrometric or spectroscopic techniques, enabling them to be

transitioned to casework. This is unprecedented and represents a significant advancement

with respect to previous work published by our group. It is, in fact, the culmination of nine

years of developments including recent protocol developments actually enabling fingermarks

enhanced at crime scene or at crime labs to be recovered and prepared for MALDI MS based

analyses.

Up to this point, methods designed and refined in laboratory settings have been proposed to

detect a large number of forensically relevant substances, such as those employing Ion

Mobility Separation for the detection of drugs and metabolites10, and/or to map them directly

on the fingermark ridges thus potentially providing the link between the biometric

information and corpus delicti11,12. Potential was also shown to detect and visualise

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to use MALDI MSI to separate overlapping ridge impressions16 and to integrate it with other

spectroscopic techniques12. This body of work has opened up an avenue to what these authors

have named "chemical criminal profiling", that is, the opportunity to infer information on

suspects' lifestyle and identity through the chemical species detected in their fingermark

residue.

The development of MALDI MSP and MSI has come a long way since their first reported use

for the analysis of latent fingermarks in 20093 and, already in 2011, there was a drive to

transition these methods to the field. One study reported on an alternative method to powder

development, one of the most common forensic enhancement techniques (FET) used by crime scene investigators (CSI), to enable visualisation and MALDI MSP/MSI analysis of

fingermarks17, thus demonstrating the operational capability of these techniques/methods.

However, arguably, current FET will still be applied to fingermarks in the first instance for

many years to come; therefore, subsequent studies investigated the compatibility between

FET and MALDI MSP/MSI ("tandem analysis") to generate a workflow which showed a

promising potential for translation into the field18.

On occasion, samples were obtained from actual crime scenes and methods were applied

crudely as developed in the laboratory19; though successful, these "one off" method

application to real crime scene marks had not been further refined. The vast majority of the

marks successfully analysed in tandem were mock samples prepared in laboratory

settings18,20. Whilst operational feasibility has now been more largely investigated with a

clear opportunity for implementation in the field, only limited predictions can be made on

methods' robustness and versatility in operational circumstances. In the authors' view, given

the extreme variability of fingermark donors’ characteristics and of the environmental

conditions the marks could be exposed to, it will take the analysis of a large number of actual

crime scene marks to make a suitable evaluation of this particular aspect.

Recently, a study was published covering investigations undertaken under the Home Office

Innovation Fund, enabling a collaboration between Sheffield Hallam University's Fingermark

Research Group (FRG) and West Yorkshire Police (WYP), UK21. These investigations

specifically concerned the development of MALDI MSP and MSI methodology for

operational use. From this collaboration, a step forward has been taken by proposing a

method to pinpoint the best sample treatment for marks enhanced using the powders most

often employed by WYP (carbon black, aluminium and TiO2); by "best sample treatment, it

is meant the treatment enabling the most chemical information and ridge detail to be obtained

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application in combination with the use of the initial enhancing forensic powder. This is

especially important for secondary lifts, which are the residual mark material lifted after a

first cycle of enhancement and tape lifting (primary lift). Secondary lifts may be all it is

granted to analyse by the Police as primary lifts are normally kept as evidence; therefore

establishing the most efficient sample treatment prior to analysis was paramount in order to

avoid loss of information, which is already minute in the remaining residue. The report

indicated suitability of any of the trialed sample preparation methodologies (except the

Dry-Wet method17) for marks preliminarily enhanced using aluminium or carbon powders,

whereas in the case of preliminary TiO2 powder enhanced marks, re-enhancing the mark

using the initial powder and spray-coating with the MALDI matrix was recommended.

Contextually, the report also demonstrated and highlighted the importance to analyse primary

lifts instead, which were demonstrated to outperform secondary lifts in terms of maximizing

forensic opportunities; in the case of primary lifts, MALDI matrix spray coating and matrix

spotting are the sample preparation methods to be applied prior to imaging and profiling

analyses respectively.

The natural evolution of the study briefly described above was the investigation of the

translational feasibility to real crime scene samples. As illustrated further in this paper, in

preparation for the analysis of real forensic evidence, preliminary consideration (followed by

protocol development) was given to the collection, transport and storage of the crime scene

marks under operational conditions; this also represents an advancement compared to

previously published work and it is a significant one as lack of compliance with CSI

requirements prevents any analysis carried out using new and developing technologies. For

primary lifts, normally a CSI would seal them in acetate backing sheets for transport and

storage of the evidence. Therefore a prior study was undertaken to assess the possibility to

obtain both ridge detail and chemical information from fingermark lifts removed from acetate

sheets which proved to be successful (data not shown). In the event that: (a) only secondary

lifts were accessible, (b) the mark had been suitably photographed and the photo kept as

primary evidence by the police or (c) the primary lifted mark was deemed to have no

evidential value by the CSI who granted access to the mass spectrometrist, a system was

devised to transport and store the lifts in a non contactless manner, avoiding contamination

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Once a way to store and transport the evidence was established, police casework could be

investigated. In particular, four examples of casework are reported here to illustrate the first

and more in depth insights into the limitations and benefits of applying MALDI MSP and

MSI under operational conditions. Given that the benefits of analysing primary lifts was

demonstrated18, the authors were granted the opportunity to only analyse primary lifted marks

or marks directly analysed on the surface of deposition.

In particular, two marks (i) and (ii) were originally located on a textured light frame and a

plug socket respectively; they were stored in the customised box (Fig S1) and subsequently

analysed as primary lifts. Another mark (iii) was analysed directly on the original surface of

deposition, a drug packet, following initial development by the crime lab. The last mark

discussed in this paper (iv) was located on the interior side of a window frame and was tape

lifted at the crime scene following enhancement with carbon black powder before being

sealed in acetate for storage.

The marks under consideration in this paper are classed as belonging to the high profile

crimes category; marks (i)-(ii) were collected at a cannabis farm during an investigation of

drug dealing, while mark (iii) was found at a crime scene of a murder; finally mark (iv) was

from an harassment case.

Experimental

Materials

Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA) and ALUGRAM1

SIL G/UV254 pre-coated aluminium slides were purchased from Sigma Aldrich (Poole, UK).

Standards of cocaine (COC), benzoylecgonine (BZE), norcocaine (NCOC), ecgonine methyl

ester (EME) and cocaethylene (CE) were provided by Kings College London (London, UK)

through of an ongoing scientific collaboration. Acetonitrile (ACN) and acetone were obtained

from Fisher Scientific (Loughborough, UK). MALDI target OPTI TOF spotless inserts were

purchased from Applied Biosystems (Foster City, CA, USA). Double-sided conductive

carbon tape was purchased from TAAB (Aldermaston, UK). Fingerprint brushes were

purchased from Tetra Scene of Crime Ltd (Essex, UK). All fingermark development powders

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Instrumentation and Instrumental Settings

Mass spectrometric analyses in positive mode were conducted using either a modified

Applied Biosystems API "Q-Star" Pulsar i hybrid quadrupole time-of-flight (QTOF) instrument (Concord, Ontario, Canada) or a Synapt™ G2 HDMS ion mobility mass

spectrometer (Waters Corporation, Manchester, UK). On the Q-star instrument, the

orthogonal MALDI source has been modified to incorporate a SPOT 10 kHz Nd:YVO4

solid-state laser (Elforlight Ltd., Daventry, UK)22, operating at 1 kHz, having a wavelength of

355 nm and a pulse duration of 1.5 ns. Images were acquired at a spatial resolution of 150 x 150 μm in raster imaging mode, using ‘oMALDI Server 5.1’ software supplied by MDS

Sciex (Concord, Ontario, Canada). MALDI MSI acquisition was performed in positive ion

mode in the mass range between m/z 50 and 1000. The declustering potential 2 was set at 25

arbitrary units, with an accumulation time of 0.117 min. The total run time varied between

60-120 minutes depending on the size of the fingermark.

The Synapt™ G2 HDMS instrument is equipped with an Nd:YAG laser which was also

operated at 1 kHz. All MS spectra were acquired in positive ion 'sensitivity' mode in the mass

range between m/z 100–1000. For MALDI-IMS-MS/MS analyses, collision induced

dissociation (CID) was performed in the T-wave™ transfer cell following initial ion mobility

separation. This allowed for all product ions to retain the same ion drift time as their

corresponding precursor ion. Collision energy was applied at 26 eV for cocaine and each of

the metabolites. In all instances, the MALDI source of the instrument was thoroughly cleaned

prior to analyses ensuring no signal arising from potentially forensically relevant molecules were detected in a subsequent blank analysis; furthermore the crime scene samples were

analysed before any standards which were used for mass spectral comparison purposes,

where appropriate. This ensured that there was no sample carryover which could falsely

indicate the presence of the substance of forensic significance in the crime scene samples.

Prior to analyses, calibration of the instrument was performed in the range between m/z

50-1000 mass range using Phosphorus Red.

A video spectral comparator (VSC4CX, Foster & Freeman, Evesham, UK) was employed

for mark (i-ii) and (iv) to take optical images as a reference for MALDI MSI analyses.

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Data processing

MALDI MS images were processed using Biomap software (Novartis, Basel). All images

were normalised against the total ion count (TIC) and specific m/z values were visualised

with optimal contrast and intensity to allow for visualisation of the fingermark ridges. Mass

spectra were processed using MassLynx 4.1 (Waters Corporation, Manchester, UK), Analyst

MDS Sciex (Concord, Ontario, Canada) and the open source multifunctional mass

spectrometry software mMass23,24.

Collection of fingermarks from crime scenes

Fingermarks (i) and (ii) were recovered from a seized cannabis farm. Visual examination

revealed (i)on a textured metal light fitting. Enhancement was performed using TiO2 powder.

Mark (ii) was visualised on a plug socket switch following enhancement using aluminium

powder. These marks were secured within the storage box shown in Fig. 1 and analysed by

MALDI MSI as primary lifts .

Mark (iii) was enhanced on a drug packet. Treatment using cyanoacrylate fuming followed

by BY40 and image capture were performed according to the Home Office Fingermark

Visualisation Manual9. The fingermark was analysed via MALDI MSI directly on the surface

of deposition without any lifting.

Finally, mark (iv) was recovered as a primary lift from an interior window frame using

forensic lifting tape following initial development using carbon black powder. The mark was

sealed in acetate prior to analysis and analysed after 18 days of storage.

Matrix deposition

For MALDI MSI experiments, fingermarks recovered from crime scenes were spray-coated with α-CHCA using a ‘SunCollect’ autosprayer (SunchromGmbH, Friedrichsdorf, Germany).

A solution of 5 mg mL-1 α-CHCA in 70:30 ACN: 0.5% TFA was sprayed onto each of the

fingermarks for a total of 4 layers at a rate of 2 μL min-1, using a ‘‘slow’’ raster setting.

For post-image MALDI MSP and MS/MS analyses, fingermarks were spotted numerous

times at different locations (0.5 μL per spot), to generate internal replicates, with the same

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areas of the ridges where the imaging analysis had indicated the presence of components of

interest.

Results and Discussion

In the study presented here, four examples of the police casework involving fingermarks as

main source of forensic evidence are reported. To date, seven casework examples have been

investigated through the application of MALDI MSI and MALDI MS based techniques to

crime scene marks, but only four will be discussed here as being the most representative of

the limitations and benefits of this technology and the methodologies developed. However, a

complete evaluation of these aspects can only be undertaken upon the analysis of a large

number of crime scene marks. This is due to the extreme variability in the state of the mark

such as "quality of the donor"25, time since deposition, surface of deposition, environmental

factors and prior forensic enhancement treatments; all of these factors can also inter-play and

influence directly the result of the application of MALDI MS based methods. Therefore these

four case studies are to be considered only a first (though significant) insight into the

feasibility and operational character of MALDI MS based methods. Following previous

published work, factually demonstrating for the first time the benefits of analysing primary

lifted marks as opposed to secondary lifted marks21, the crime lab and the CSI sent for

analysis marks that had been collected as primary lifts in three instances and, in one case, the

actual specimen on which the mark was deposited. The four marks were all subjected to

MALDI MSI analysis and in some instances to further mass spectrometric characterisation

through Tandem MS in order to identify the molecules detected.

Marks (i) and (ii) were collected as primary lifts after enhancement with TiO2 and aluminium

powder respectively. MALDI MSI analyses were conducted after 1 and 2 days from the

collection and the results are shown in Figure 2. Prior to enhancement by the CSI, an orange

colouration was observed within the ridges of mark (i) (Fig 2A); unfortunately it was not

possible to ascertain if the orange compound was pre-existing on the surface, deposited

together with the mark or the result of a coincidental association (landed on the mark at a

later stage following mark deposition). Further enhancement with TiO2 powder did not yield

any useful ridge detail. The primary lift exhibited a poor amount of material by visual

inspection and even the faintest hint of ridge detail, observed when the mark was still on the

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mark being strongly adhered to the surface, or a very old mark. The lifted mark was in any

case subjected to MALDI MSI analysis. The analysis confirmed scarcity of fingermark

residue material. No ion images of any minutiae could be retrieved and Fig 2C shows a

representative example; this could also be ascribed to the surface being textured, which is an

objective problem for conventional enhancement techniques too, due to physical distortion

and interruption of the ridge pattern. Also, no useful molecular information could be obtained.

It could be speculated that the mark had probably been subjected to high temperature on the

light fitting, thus causing molecular thermal degradation. This is an example illustrating the

limitations that the MALDI MS based methods may encounter with poor lifts and scarcity of molecular information, possibly due to severely aggressive environmental factors and age.

The initial enhancement of mark (ii) was also poor and the CSI deemed the very little ridge

detail recovered insufficient for an identification. The mark was therefore lifted, stored in the

customised box and transported for mass spectrometric analysis. The acetate was peeled off

and an optical image of the lift was taken before and after matrix deposition using a video

spectral comparator. Although the ridge detail could not be improved in terms of quantity or

quality upon MALDI MSI, probably due to an objective lack of ridge details, molecular

information could be this time retrieved (Fig 3); in particular, endogenous species such as

fatty acids, diacyl and tryacylglycerols were detected and identified on the basis of previous

work3,25 (data not shown). Figure 3A shows the molecular image of oleic acid at m/z 283.2 as

example of this class of molecules. Interestingly, also a previously detected substance12,16 was

observed here at m/z 304.2. and 332.2 and these ions were used to generate molecular images

of the mark (Fig 3B). These ions correspond to n-alkyl dimethylbenzylammonium (DMA)

ions having 12 and 14 CH2 repetition units respectively. n-alkyl DMA is a known

antibacterial agent found in toiletry products, wipes and detergents. This is not a forensically

relevant substance. However its detection in operational conditions indicates feasibility of

application of MALDI MS based methods for real crime scene marks and therefore it was

important to confirm its presence. For this purpose, MALDI MS/MS analyses were

performed. Interestingly, not only was the n-alkyl DMA presence confirmed through its ion

fragment at m/z 212.3, but also the presence of cocaine could be ascertained through the

detection of its characteristic ion fragment at m/z 182.2 (Fig 3C). DMA (12CH2)

(monoisotopic m/z 304.3004) and cocaine (monoisotopic m/z 304.1548) have in fact the

same nominal mass; therefore during the MS/MS analysis, the quadrupole in the QTOF

instrument would have selected both ions for fragmentation, subsequently yielding ion

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Cocaine is a forensically relevant substance and although in this case its detection may not

significantly contribute to the investigation, it does again indicate the operational character of

the technologies/methodologies employed. Initially, it seemed puzzling that, while cocaine

was detected, THC was not, despite the mark was retrieved in a seized cannabis farm.

The absence of detection could fit within previously encountered instances in which the

ionisation efficiency of this molecule in latent marks is poor compared to other drugs of

abuse analysed.

Recently a derivatisation method to detect and image THC in hair has been proposed26 and

work is in progress to trial and apply this method to THC in fingermarks.

There is also a possibility that THC could have degraded into cannabinol and/or cannabidiol

as it is found in aged marijuana samples and it is reasonable to think that heat and light

accelerates the aging. However, no evidence for the presence of these species was found by

examining the data acquired in MS mode. Detection of these species though, is not to be

excluded, if a transfer fragmentation experiment, using an Ion Mobility Mass Spectrometer

and the relevant standards had been carried out (as for Mark (iv) further discussed in this

paper).

However, presence of THC in the mark, is not necessarily to be expected, even in a cannabis

farm. It is in fact highly likely that gloves were used during the manufacturing process, as the

frequent retrieval of "marigold" at these scenes indicates; therefore the presence of cocaine

could be the result of coincidental and later association.

Mark (iii) was located on a drug packet and the evidence was acquired within a murder case.

At the crime lab, cyanoacrylate fuming followed by BY40 was employed to enhance the

mark which yielded poor ridge detail (Fig 4A). The mark was subsequently sent to be

subjected to MALDI MSI in the effort to obtain additional ridge detail. No lifting was

performed as the mark could be analysed directly on the drug packet due to the surface being

flat and thin. The analysis was performed after one month from the collection. Only one ion

provided a degree of ridge pattern reconstruction at m/z 104.0 (suspected choline) but the

total ion current selection in the m/z range 100-1000 provided more ridge pattern coverage.

The choline MS image provided a lower quality image than that obtained by Cyanoacrylate

fuming/BY40 while the total ion current image rivalled the amount of ridge detail obtained

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Although disappointing, these results are not surprising. While it has been shown that

MALDI MSI can be successfully applied to cyanoacrylate fumed marks, the subsequent

treatment with BY40 greatly suppresses ionisation20; this is probably due to the ability of this

dye to sequestrate the energy from the laser, thus rendering the MALDI process ineffective

for the other molecules present. In previously published work20, it was shown that while THC

and Amphetamine could not yield ridge detail following cyanoacrylate fuming/BY40

treatment, cocaine and heroin provided useful molecular images of the ridge pattern. In that

study, fingermarks deposited on aluminium slides were produced after spiking fingertips

using a 50 µL aliquot of mixed drug standard solution (4 drugs in total) equivalent to 1.25 µg of pure cocaine and of pure heroin spread over the mark surface. Therefore within this

casework, either these drugs were not present in the mark or their amounts were lower than

the instrumental detection limit. The MALDI analyst was never told the content of the drug

packet and therefore it is difficult to comment beyond these hypotheses.

Casework (iv) was the one that highlighted the most the operational character of the

technology developed as well as the complementarity of the intelligence that it is possible to

obtain and that can contribute to the overall investigations and court cases. This was an

harassment case in which a mark was located on the interior of a window frame. The initial

optical examination of the latent fingermark sample showed that it had an oily consistency, similar to what may be observed from a “groomed”5

fingermark. The CSI deemed it

appropriate to use carbon black powder to enhance the mark prior to lifting it.

Unfortunately, the result of this enhancement was not conducive to the retrieval of useful

ridge detail (Fig 5A); the primary lift, which was sealed in acetate, subsequently subjected to

MALDI MSI analysis, aiming to the retrieval of more minutiae.

However, the initial observation of the enhanced mark did not show potential for

improvement; as for any other technique, ridge detail cannot be generated if it is not

originally present. The ridge pattern on the lift appeared to be very smudged. This could be

the result of either of the following circumstance or combination thereof 1) enhancement, 2)

merging of the ridges upon recovery of an oily mark which was then compressed on acetate

3) original state of the mark after deposition. The MALDI MS imaging analysis confirmed

the initial concern as there was no improvement of the ridge detail. Figures 4B and C show

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While these images were not useful in terms of physical information, they were obtained

exploiting the detection of cocaine which, in its intact form, exhibits a m/z at 304.1 as well as

of a fragment (in source fragment) at m/z 182.1.

Therefore, once again, cocaine was detected within a mark in operational conditions. Its

presence was also confirmed by conventional investigation of the detectives who meanwhile

interrogated the suspect. The suspect confessed their crime and was subsequently subjected to

a drug test which confirmed cocaine abuse.

Therefore while the MALDI MSI analysis could not be definitive about drug consumption, as

the presence of the intact molecule of cocaine in the mark cannot discriminate between drug handling and drug consumption, ordinary investigation could confirm drug abuse.

However, the MALDI analyst was blind to all the details of the casework and of the results of

the ongoing investigations and decided to conduct a more in depth analysis searching for

metabolites which could prove drug abuse.

It has been previously demonstrated that the use of Ion Mobility Mass Spectrometry

separation tandem Mass Spectrometry MALDI-IMS-MS/MS, enables a sensitive and reliable

detection of drug metabolites, distinguishing those sharing the same nominal mass on the

basis of their mobility1, using transfer fragmentation experiments.

In this type of analysis, the ion is subjected to ion mobility first and then fragmentation in the

transfer cell of the triwaveTM region of the instrument in order for the generated ion

fragments to retain the same drift time as their parent ion.

Prior to this analysis the MALDI matrix was preliminarily spotted in 3 different locations of

the mark and outside the mark in order to ensure that any detected and forensically relevant

species were real and also not present in the matrix.

Transfer fragmentation MALDI IMS-MS/MS analysis was then performed out in these

locations first followed by the same analysis applied on standards (and not the other way

around to avoid carry over effects in the instrument). The ions preliminarily detected were

previously encountered within the drug/metabolite analyses in fingermarks performed by

these authors and therefore standards were available to confirm identity. Transfer

fragmentation analysis was employed here and confirmed the presence of cocaine (theoretical

and experimental m/z 304.1549 and 304.1737respectively) as well as revealing the presence

of benzoylecgonine (theoretical and experimental m/z 290.1392 and 290.1240 respectively),

ecgonine methylester (theoretical and experimental m/z 200.1287 and 200.1551respectively),

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Additionally another interesting ion was revealed, having a m/z at 318.1158 which was not

previously detected within MALDI MSI analysis on the less sensitive instrument (MALDI

Q-TOF Qstar Pulsar i, Applied Biosystems). MALDI IMS-MS/MS analysis of both the mark

and the standard (Fig 6) allowed for Drift Scope Plots (a graphical representation of the Ion

Mobility Separation) to be generated and select DT regions of interest containing both the

parent and the corresponding product ions (Fig. 6A). Spectra from these acquisitions were

interrogated for a product ion at m/z 196.1481 (Fig 6B) derived from the collision induced

dissociation of the ion at m/z 318.1974 which directly corresponded to those observed in the

standard . Drift time chromatograms could then be produced based on the specific DT of this product ion, showing that the product ion in the standard and the mark directly coincided

having the same drift time (Fig. 6C). This ion peak was also absent from the matrix blank

which indicated that it did not arise from it. This analysis confirmed the identity of this ion as

cocaethylene (theoretical m/z 318.1705). This metabolite only forms in the body when

cocaine is consumed with alcohol27. This information could be very important both in

investigations and in a court case as it highlights the state of mind of the individual at the

time of committing the crime. While these analyses were being performed, the investigators

questioned the individual about alcohol consumption and he firmly denied this instance. Only

much later, prior to the case being discussed in Court he confessed to have also been drinking

in addition to cocaine consumption. This confirms and highlights once more benefits (current

and potential), importance and operational character of the developed methodologies for

profiling a suspect, their lifestyle or the circumstances of the case from a mark.

Particularly casework example (iv) represents a significant advancement with respect to the

work previously published by this group. Transfer fragmentation experiments on an Ion

Mobility Mass Spectrometer were already successfully reported, demonstrating the

opportunity to detect THC, methadone and cocaine's primary metabolite benzoylecgonine10.

However, in that case, a mark was taken "fresh" in controlled conditions (environmental,

ageing and collection wise) from a patient in a rehabilitation clinic. As opposed to mark (iv),

the mark analysed in Bailey et al10 remained undeveloped (no FET were applied), thus

avoiding any chemical interference causing ion suppression. Also, differently from the work

presented here, in Bailey et al10 the authors carried out a targeted analysis searching for

"commissioned" molecular targets. Finally, differently from the example presented in Bailey

et al10, in the casework example presented here, transfer fragmentation experiments were

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highlights the sensitivity of the technology, given that some material would have been ablated

already through the MALDI MSI analysis.

A mention must be made to the robustness of these protocols. Mark (iv) was very

successfully analysed 17 days after collection yielding important molecular information as

well as molecular images. Although mark (i) analysed after one day from collection did not

yield any information, this could be due to a number of environmental reasons and/or to

inefficient lifting. On the contrary, the analysis of mark (iv) indicates the possibility to

"re-visit" crime scene exhibits but may also indicate the opportunity to analyse cold cases involving fingermarks; using this technology, this opportunity was recently demonstrated for

a 9 year old blood palm print preliminarily enhanced with acid black 128 and for a 30 year old

stain on fabric which was preliminarily enhanced with ninhydrin (data not shown).

Conclusions

For the first time, crime scene marks from high profile cases were analysed in operational

conditions by MALDI MS based methodologies. These marks were analysed as primary lifts

or directly from the surface of deposition following enhancement by a conventional forensic

technique (FET) as deemed appropriate by the CSI or the Crime Lab investigator. This paper

also reports on the opportunity to cover the analysis of marks stored in acetate (rotine CSI

practice), thus increasing the range of type of collected marks that is possible to investigate

with these methodologies. Together with the intelligence that it was possible to retrieve, this

highlights the improved value scientific of this work, with respect to published work, through

the demonstration of a much higher potential to impact forensic practice, forensic

investigations and judicial debates. The opportunity to apply MALDI MS based methods,

tailored for the analysis of fingermarks within police casework has enabled a preliminary

evaluation of the operational feasibility of the methods themselves. The word "preliminary"

is very appropriate due to the unpredictable nature of the state of the crime scene mark

coupled to a prior physical/chemical treatments. As conditions cannot be controlled like for

mock samples prepared in the lab, these factors, together with the unknown but certainly

minute amounts of forensically relevant substances in fingermarks, could have well pointed

out to inapplicability of MALDI technology/developed methods for the retrieval of chemical

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ridge detail was inferior or only rivalling that provided by FET, overall the nature of the

intelligence retrieved in operational conditions is useful to chemical profiling the suspect,

their state of mind or the circumstances of the crime; this is important in order to steer

investigations and/or to contribute towards court case debates. Even the instances in which

the detection of cocaine could not significantly contribute to the case, this report

demonstrates robustness of the MALDI methods developed to date to stand operational

conditions and integrate with the prior application of FET. In all cases, and on the basis of

previous developmental work, hypotheses could be made on the quality of the performance of

this technology indicating limitations (either deriving from the samples itself or due to the nature of the techniques) as well as benefits. Both in terms of limitations and benefits, this

study must be considered only a first insight on the potential of this technology to be used in

the field. In order to evaluate its robustness and feasibility fully, a large number of crime

scene marks will need to be analysed to cover the wide range of factors making fingermarks

an extremely variable specimen in terms of the physical and chemical information that it is

possible to retrieve.

Acknowledgments

Fingerprint expert John Dixon from West Yorkshire Police is gratefully acknowledged for his

concept and making of the latent fingermark storage and transportation box. This work was

funded by the Home Office Innovation Fund [2014], UK.

References

1 D.R. Ifa, N.E. Manicke, A.L. Dill, R.G. Cooks, Science, 2008, 321,805. 2. F. Rowell, K. Hudson, J. Seviour, Analyst, 2009, 134, 701

3. R. Wolstenholme, R. Bradshaw, M.R. Clench and S. Francese, Rapid Commun. Mass

Spectrom. 2009, 23, 3031.

4. M. I. Szynkowska, K. Czerski, J. Rogowski, T. Paryjczak, A. Parczewski, Surf. Interface Anal, 2010, 42, 393

5. R.A. Musah, R.B. Cody, A.J. Dane, Angela L. Vuong, J.R.E. Shepard, Rapid Commun.

(17)

16

6. A. Tripathi, E.D. Emmons, P.G. Wilcox, J.A. Guicheteau, D.K. Emge, S.D. Christensen,

and A.W. Fountain III, Appl. Spectros., 2011, 65, 611

7. K.M. Antoine, B.S. Shirin Mortazavi, A.D. Miller, and L.M. Miller. J Forensic Sci, 2010,

55, 513

8. Ricci C, Phiriyavityopas P, Curum N, Chan KLA, Jickells S, Kazarian SG. Appl. Spectros.

2007, 61: 514

9. H. Bandey, V. Bowman, S. Bleay, R. Downham and V.H. Sears, Fingermark Visualisation

Manual, ed. H. Bandey, CAST, Home Office, Sandridge, UK, 2014.

10. M.J. Bailey, R. Bradshaw, S. Francese, T.L. Salter, C. Costa, M. Ismail, R. Webb, I. Bosman, K. Wolff and M. de Puit, Analyst, 2015, 396, 6254.

11. R. Bradshaw, R. Wolstenholme, R. Blackledge, M.R. Clench, L.S. Ferguson and S.

Francese, Rapid Commun. Mass Spectrom., 2011, 25, 415.

12. R. Bradshaw, R. Wolstenholme, L.S. Ferguson, C. Sammon, K. Mader, E. Claude, R.

Blackledge, M.R. Clench and S. Francese, Analyst, 2013, 138, 2546.

13. K. Kaplan-Sandquist, M.A. LeBeau, M.L. Miller, Forensic. Sci. Int., 2014, 235, 68. 14. K. Kaplan-Sandquist, M.A. LeBeau, M.L. Miller, J. Forensic Sci., 2015, 60, 611. 15. L. Sundar, F. Rowell, Analyst, 2014, 139, 633.

16. R. Bradshaw, S. Bleay, R. Wolstenholme, M.R. Clench and S. Francese, Forensic Sci

Int., 2012, 222, 318.

17. L. Ferguson, R. Bradshaw, R. Wolstenholme, M.R. Clench and S. Francese, Anal.

Chem., 2011, 83, 5585.

18. R. Bradshaw, S. Bleay, R. Wolstenholme, M.R. Clench and S. Francese S., Forensic Sci.

Int. 2013, 232, 111.

19. R. Bradshaw and S. Francese, Spectroscopy Europe,

http://212.113.150.109/articles/55-

articles/3357-matrixassisted-laser-desorption-ionisation-tandem-mass-spectrometry-imaging-of-small-molecules-from-latent-fingermarks, 2014, accessed 6/02/2017.

20. G. Groeneveld, M.dePuit, S. Bleay, R. Bradshaw and S. Francese, Scientific Reports,

2015, 5,11716.

21. R. Bradshaw, N. Denison and S. Francese, Anal. Methods 2016, 8, 6795.

22. P.J. Trim, M.C. Djidja, S.J. Atkinson, K. Oakes, L.M. Cole, D.M.G. Anderson, P.J. Hart,

S. Francese and M.R. Clench Anal. Bioanal. Chem., 2010, 397, 3409.

23. M. Strohalm, M. Hassman, B. Kosata and M. Kodicek, Rapid Commun. Mass Spectrom.,

22, 2008, 905.

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17

25. L.S. Ferguson, S. Creasey, R. Wolstenholme, M.R. Clench and S. Francese, J Mass

Spectrom,. 2013, 48, 677.

26. E. Beasley, S. Francese and T., Bassindale, Anal. Chem., 2016, 20, 10328.

20. M.R. Brezinski, B.J. Spink, R.A. Dean, C.E. Berkman, J.R. Cashman and W.F. Bosron,

Drug metabolism and disposition. 1997, 25, 1089.

28. E. Patel, P. Cicatiello, L. Deininger, M. R. Clench, G. Marino, P. Giardina, G.

Langenburg, A. West, P. Marshall, V. Sears and S. FranceseAnalyst, 2016, 141, 191.

.Legends

Figure 1. Customised storage and transportation box. The box accomodates primary/secondary lifts with or without acetate backing sheet. Pins will secure the lift and an

acetate sheet is inserted in the inside wall of the box lid to protect from debris the fingermark

lifts that are not placed on acetate sheets.

Figure 2. MALDI MSI analysis of a primary fingermark lift recovered on a textured light frame at a seized cannabis farm. (A) and (B) show the optical images of the fingermark on

the surface as observed with no treatment and of the primary lift after enhancement with TiO2

respectively; (C) shows the molecular image of an unidentified ion at m/z 343.0 which is

representative of the scarce quality of the data obtained; no useful molecular information of

ion image showing ridge detail could be obtained.

Figure 3. MALDI MSI analysis of a primary fingermark lift recovered on a textured light plug socket at a seized cannabis farm. (A) shows the optical image of the primary lift after

enhancement with aluminium powder; (B) shows the molecular distribution of oleic acid at

m/z 283.2; (C)-(D) show the molecular images of two ions at m/z 304.2 and 332.2,

normalised against the matrix peak at m/z 190.0, which were tentatively assigned to n-alkyl

dimethylbenzylammonium ion (DMA), with the possibility of the former ion actually being cocaine, and with the latter ion carrying two extra CH2 units for DMA. The MS/MS spectrum

in panel E confirms the presence of both DMA and cocaine which share the same molecular

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Figure 4. MALDI MSI analysis of a fingermark enhanced with cyanocrylate fuming following by BY40 on a drug packet. (A) shows the optical image of the fingermark after

chemical enhancement; (B) and (C) show the molecular distribution of an ion at m/z 104.0,

normalised against the matrix peak at m/z 190.0, and of the total ion current over the m/z

100-1000 which best reconstruct the ridge pattern; the ion at m/z 104.0 has been tentatively

assigned to choline whereas the latter remains identified. However their identity is

unimportant in this context.

Figure 5. MALDI MSI analysis of a primary fingermark lift recovered on the interior of a window frame following enhancement with carbon black powder. (A) shows the optical

image of the enhanced mark; (B) and (C) show the MALDI MS images of two ions at m/z

304.1 and 182.1, normalised against the matrix peak at m/z 190.0, which were assigned

tentatively to cocaine and its in source ion fragment respectively; these ions were later

confirmed using by transfer fragmentation analysis.

Figure 6. MALDI-IMS-MS/MS analysis of the ion at m/z 317.1158 suspected to be cocaethylene (COCE) within a primary fingermark lift recovered on the interior of a window

frame following enhancement with carbon black powder. Panel A shows the drift scope plots

of COCE standard (i), COCE in 3 location of the marks (ii-iv) and 1 location outside the

mark (matrix, (v)) that were generated through transfer fragmentation experiments. Panel B

shows the MS/MS spectra of the ion at m/z in the COCE standard and in the 4 locations (3 in

the mark and one outside it) (vi-x) after the selection of the parent ion and products ion

within the same drift time plume in the fingermark and in the standard. Selection of the ion

product at m/z 196.1481 in common to the COCE standard and COCE in fingermark and

References

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